In recent years, the need for high throughput strategies for the analysis of biological samples has increased dramatically. One area of development is the design of high throughput techniques for the analysis of nucleic acids. Analysis of nucleic acids frequently requires that they be separated from various impurities generated during synthesis or isolation.
A common impurity in a sample of nucleic acids is salt. Many analytic and diagnostic assays of nucleic acids require that salts be removed from nucleic acid samples prior to analysis. Often times, this necessitates one or more separation and/or purification steps in order to desalt the sample comprising the nucleic acid.
One analytic technique which often requires desalting of nucleic acid samples is matrix-assisted laser desorption-ionization-time-of-flight (MALDI-TOF) mass spectrometry (MS) (MALDI-TOF MS) (see, e.g., Griffin, T. J. and Smith, L. M., Trends Biotechnol. 18:77-84 (2000)). When used to analyze nucleic acids, MALDI-TOF MS involves laser-induced desorption and ionization of nucleic acid molecules which are embedded in a large excess of a crystalline matrix. Laser-induced desorption and ionization of nucleic acid molecules results in production of ions that acquire the same initial energy when accelerated in an electric field, and allows for separation of nucleic acids based on mass-to-charge ratios when performed in high vacuum.
Desalting of nucleic acid samples is necessary for MALDI-TOF, MS analysis of nucleic acids because the positive ions (cations) of a salt can interact with the negatively-charged sugar phosphate backbone of the nucleic acid, resulting in signal dilution and complicating data analysis.
Several methods for desalting nucleic acid samples are currently available, including dialysis, solid phase extraction techniques, size exclusion filtration techniques, affinity capture techniques including magnetic bead binding and washing, ethanol precipitation and ion exchange. With the exception of ion exchange, these desalting methods all require one or more washing and/or separation steps in order to remove the salt from the nucleic acid. Moreover, all of these desalting methods, with the exception of ion exchange, suffer from complications when subjected to high-throughput analysis. For example, magnetic bead desalting can be difficult to automate, solid phase extraction techniques can suffer from cross talk when used in a 96- or 384-well format, and size exclusion filtration techniques can result in the loss of sample during filtration and/or resuspension steps. Further, these techniques require movement of sample plates to and from filter stations and/or specialized equipment, which can be difficult and costly to fully automate.
While ion exchange can eliminate washing and/or separation steps and is suitable for high-throughput analysis, there are other problems associated with the use of ion exchange to desalt nucleic acids. For example, gel-based ion exchangers have typically been used to desalt nucleic acids. However, gel-based ion exchangers can absorb liquid, as well as ions. For dilute solutions of nucleic acids, this results in the trapping of nucleic acids in the gel-based ion exchangers, thereby severely hampering analysis. Thus, the use of gel-based ion exchangers for desalting nucleic acids can give inconsistent results.
A need exists for a method of desalting samples comprising nucleic acids that overcomes the limitations and problems of the current methods and further allows for efficient high-throughput analysis while avoiding time-consuming and laborious conventional desalting steps.